UCSF

Genetically engineered therapeutic cells are a new paradigm in treating historically intractable diseases. Chimeric antigen receptor (‘CAR’) T cells have made a tremendous impact in the clinic against blood cancers, but potent signaling through the CAR can result in toxicity and CAR T cell exhaustion. Further engineering efforts to modulate CAR signaling are needed to continue to deploy engineered cell therapies against increasingly complex maladies. The use of protein degradation to ablate CAR signaling is a promising method for improving CAR T cell safety and efficacy. In preclinical models, small molecule-controlled protein degradation to tightly control CAR expression levels was shown to circumvent issues of CAR T cell toxicity and exhaustion. An equally important complement to small molecule modes of control are genetic circuits that allow therapeutic cells to reactively modulate CAR signaling in response to their environment. Using genetic circuits in combination with protein degradation could result in the birth of a new class of engineered cell therapies capable of cell autonomous regulation of therapeutic function. However, programmable targeted protein degradation tools for this purpose do not exist for immune cell engineering. To address this gap in the existing CAR T cell engineering toolbox, we designed a novel bioPROTAC that uses two protein domains to bridge a target protein of interest with machinery from the ubiquitin proteasome system to promote proteasomal recruitment and degradation. We built the bioPROTAC such that it is capable of degrading both cytosolic and membrane proteins while also being as small as 181 base pairs for efficient delivery by lentivirus. We show that the bioPROTAC is capable of degrading cytosolic proteins such as green fluorescent protein (‘GFP’) in multiple mammalian cell lines, as well as degrading and functionally modulating membrane proteins such as second-generation CARs in Jurkat and primary T cells. To demonstrate our bioPROTAC’s ability to be composed into genetic circuits that modulate cell signaling pathways, we engineer an antigen-inducible circuit capable of shutting down CAR signaling through degradation of the tyrosine kinase ZAP70. Through this circuit, we show that this new bioPROTAC can interface with and modulate endogenous signaling networks under user-defined cell autonomous control. Genetic circuits that actuate protein degradation can be a powerful method for engineering self-regulating CAR T cells thereby improving therapeutic safety and efficacy.